Calcium transient is originated from calcium concentration gradients across biological membranes and determined by the calcium-binding affinity and kinetics of calcium channels/pumps as well as intracellular calcium-binding proteins. The spatial-temporal calcium concentration change results in different physiological signal transduction, including muscle contraction, heart beating, neurotransmitter release, and gene expression, etc. (Clapham D E (2007) Cell 131: 1047-1058; Berridge et al., (1998) Nature 395: 645-648; Berridge M J (1998) Neuron 21: 13-26; Bers & Guo (2005) Ann. N. Y. Acad. Sci. 1047: 86-98; Spitzer N C (2008) Nat. Neurosci. 11: 243-244).
The time scale of calcium signaling is varied, ranged from mille-seconds to minutes. Fast calcium signaling, especially associated with action potential, usually occurs with a rapid local calcium rise (milliseconds) due to calcium influx via the membrane voltage-gated calcium channel and calcium release from internal stores, for example, excitation-contraction coupling (EC coupling) in muscle cells and neuron-transmitter release in neuron cells (Berridge M J (1998) Neuron 21: 13-26; Rios & Pizarro (1991) Physiol. Rev. 71: 849-908; Schneider M F (1994) Ann. Rev. Physiol. 56: 463-484; Baylor & Hollingworth (2003) J. Physiol. 551: 125-138; Bean B P (2007) Nat. Revs. Neurosci. 8: 451-465; Locknar et al., (2004) J. Physiol. 555: 627-635; Sandler & Barbara (1999) J. Neurosci. 19: 4325-4336; Borst & Sakmann (1999) Philosoph. Trans R. Soc. London. Series B, Biol. Sci. 354: 347-355; Lopez-Lopez et al., (1995) Science 268: 1042-1045; Cannell et al., (1995) Science 268: 1045-1049; Polakova et al., (2008) J. Physiol. 586: 3839-3854; Fill & Copello (2002) Physiol. Rev. 82: 893-922). Slower calcium signaling usually happens in cellular events such as an immune response, which can last minutes and to hours. In slow calcium signaling pathways, the calcium transient is controlled by several factors and secondary messengers like DAG, IP3 and ATP, involving more complicated regulation mechanisms.
To accurately monitor calcium transients in terms of kinetics, amplitude and duration, calcium indicators are required to have several key properties. It is necessary to match the dissociation equilibrium constant Kd of calcium indicators to the resting calcium concentration of the sub-cellular compartment in the magnitude of 102 s−1. To detect fast calcium-release from calcium pools in muscle and neuronal cells, calcium-binding affinity in the range of 0.1-1 mM and a calcium disassociation-rate of the indicator greater than 500 s−1 is necessary.
The development of genetically-encoded indicators (GECIs) allows probing spatial-temporal cellular events and cell signaling in real time. GECIs are a big family including, but not limited to, GCaMP, GECO, TN and the Cameleon series. They are composed of a fluorescent protein moiety and take advantage of the native cytosolic calcium-binding proteins (CBPs) Calmodulin (CaM) or Troponin C (TnC) to sensor calcium and calcium-induced global conformational rearrangements. Each CaM or TnC can bind four calcium ions in a cooperative manner with a high calcium-binding affinity (Kd=10−7 M) at the cytosolic calcium change and their calcium-binding on-rates are in the magnitude of 107 M−1 s−1. The high calcium-binding affinity and on-rate enable them to sense the immediate [Ca2+] rise in the cytosol.
These GECIs, however, have slow dissociation-rates of around 0.1-10 s−1 likely due to the cooperativity associated with multiple calcium-binding sites and multiple layers of conformational change. The slow kinetics of signal decay is disadvantageous to probe physiological fast calcium transient, especially in the neuron and skeletal muscle cells. Therefore, efforts have been made to reduce the calcium-binding affinities. One typical example was Cameleon D1ER, which has a multiple Kds around 0.8 and 60 μM and an off-rate of about 256 s−1. However, it is still not fast enough to capture calcium release from sarcoplasmic reticulum (SR) upon the stimulation in the mouse FDB fibers.
Accordingly, to fulfill the unmet need of a fast calcium indicator, a calcium indicator, designated “CatchER” was generated without incorporating a native calcium-binding domain by engineering a calcium-binding site into a single fluorescent protein EGFP. The calcium-binding stoichiometry is 1:1 and the Kd is 0.18 mM in vitro and 0.8 mM calibrated in situ, allowing the measurement of basal calcium in different cell lines and their changes responding to different drugs. Compared to Cameleon D1ER, CatchER exhibited faster kinetics, allowing it to catch the calcium change in SR in the skeletal muscle cells.